Systems, methods, and apparatus for differential phase contrast microscopy by transobjective differential epi-detection of forward scattered light

ABSTRACT

Systems, methods, and apparatus for differential phase contrast microscopy by transobjective differential epi-detection of forward scattered light are provided. In some embodiments, a microscope objective comprises: a housing with mounting threads at a second end; optical components defining an optical axis, comprising: an objective lens mounted at a first end, configured to collect light from a sample placed in a field of view, the plurality of optical components create a pupil plane at a first distance along the optical axis at which rays having the same angle of incidence on the objective lens converge at the same radial distance from the optical axis; a photodetector within the housing offset from the optical axis at a second distance along the optical axis; and another photodetector within the housing at second distance along the optical axis and offset from the optical axis in the opposite direction from the first photodetector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on, claims the benefit of, and claims priorityto U.S. Provisional Patent Application No. 62/796,703 filed on Jan. 25,2019, and U.S. Provisional Patent Application Ser. No. 62/892,621 filedon Aug. 28, 2019. Each of the preceding application is herebyincorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberFA9550-17-1-0277 awarded by the Department of Defense. The governmenthas certain rights in the invention.

BACKGROUND

Differential interference contrast (DIC) microscopy is a decades-oldtechnique that provides contrast in unstained samples by bringing outsubtle refractive index differences, and has been used for label-freeimaging of cells and thin biological specimens. However, DIC operates inthe transmission geometry in which light is provided from a first sideof a sample, transmitted through the sample, and impinges on a detectorplaced on the other side of the sample. DIC microscopy images phaseobjects in transparent samples by detecting small phase differences oftwo closely-spaced light paths propagating through the phase object. Ina conventional DIC microscope, the phase difference is detected.interferometrically using a detector placed in transmission geometry,opposite to the side of illumination. DIC is not compatible with imagingscattering samples such as thick biological tissues, however, becausethe phase of the transmitted light is not preserved after undergoingmultiple scattering events. In addition, placement of detectors behindthe tissue is not always possible, especially for imaging whole intactorganisms, as the light cannot penetrate through the entire thickness ofthe sample.

Differential phase contrast (DPC) scanning laser microscopy providesimages similar to DIC. but does not require polarization optics and canbe performed in tandem with other point scanning modalities such asconfocal and multiphoton microscopy. Instead of a wide-field camera, DPCemploys split detectors placed on the opposite side of the sample in aconventional transmission geometry. Accordingly, while obtaining DIC- orDPC-like images in thick biological samples would be extremely usefulfor clinical and preclinical imaging, placing an optical detector on theother side of an in vivo sample is nearly always impossible because oflimited light penetration through the scattering tissue. For years, ithas not been possible (and not for lack of trying by practitioners inthe field) to obtain DIC- or DPC-like images when the detectors areconstrained to be on the same side of the sample as the illumination.

Until recently, thick tissue imaging has been undertaken usingtechniques such as reflectance confocal microscopy (RCM) and opticalcoherence tomography (OCT) that detect directly backscattered photonswhile rejecting multiply scattered photons using a confocal pinhole inRCM, or a coherence gate in OCT.

More recently, oblique back illumination microscopy (OBM) techniqueswere developed in which a light source can be placed on the same side ofthe sample as detectors. Unlike RCM and OCT, which can detect only sharprefractive index changes and often suffer from speckle noise, OBM candetect slow refractive index variation in biological tissue, and doesnot have the same susceptibility to speckle noise. In the originallyproposed wide-field OBM implementation, the oblique back illuminationwas provided by two optical fibers arranged on the sides of an objectivelens, alternately providing illumination from the two sides that wereback scattered and captured through the objective lens to generate adifferential contrast image on a camera. In a later-proposed scanningOBM implementation, the illumination was delivered through the objectivelens, while two or more optical fibers were placed such that the face ofthe optical fiber collects light just to the side of the objective lens,to collect light from the tissue after multiple scattering, and guidethe light to the detectors to generate the differential signal. Whilesuch a system can facilitate generation of DPC-like images of in vivosamples, the optical arrangement is cumbersome, and difficult to operateconsistently as the signals from the two optical fibers must be balancedto generate the differential signal. Due at least in part to thesedifficulties, clinical implementations of scanning OBM systems usingoptical fiber detectors have not yet been realized.

Accordingly, systems, methods, and apparatus for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light are desirable.

SUMMARY

In accordance with some embodiments of the disclosed subject matter,systems, methods, and apparatus for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light are provided.

In accordance with some embodiments of the disclosed subject matter, asystem for transobjective differential epi-detection of forwardscattered light is provided, the system comprising: a scanningmicroscope comprising: a light source; an optical train defining anoptical path of the scanning microscope having an optical axiscomprising: scanning components optically coupled to the light sourceand configured to scan a beam from the light source across a surface ofa sample; and a microscope objective optically coupled to the scanningcomponents ; and a detector mechanically coupled to the scanningmicroscope along the optical path within a first distance of a pupilplane of the optical train, the detector comprising: a printed circuitboard defining a central clear aperture having a center configured tocoincide with the optical axis of the optical path; a first photodiodemechanically coupled to the printed circuit board at a first radialdistance from the center; and a second photodiode mechanically coupledto the printed circuit board at the first radial distance from thecenter and on an opposite side of the central aperture from the firstphotodiode, wherein the first distance is less than or equal to twicethe first radial distance; an amplifier electrically coupled to thedetector, comprising: a first transimpedance amplifier configured toreceive a first current signal from the first photodiode and provide afirst voltage signal as an output; a second transimpedance amplifierconfigured to receive a second current signal from the second photodiodeand provide a second voltage signal as an output; a differentialdetection amplifier configured to receive the first voltage signal andthe second voltage signal, and provide a third voltage signal indicativeof a difference between the first current signal and the second currentsignal as an output; and at least one hardware processor that isprogrammed to: cause the light source to emit a beam of light toward thesample via the optical train; cause the scanning components to scan thebeam of light across the sample; receive, from the differentialdetection amplifier, a plurality of output signals, each of theplurality of output signals indicative of a structure of the sample atlocation at which the beam was focused; generate an image based on theplurality of output signals; and cause the image to be presented using adisplay.

In some embodiments, the detector is integrated within the microscopeobjective.

In some embodiments, the detector is mounted between the microscopeobjective and the second plurality of lenses, the detector furthercomprising: a housing supporting the printed circuit board; firstthreads configured to receive the microscope objective; and secondthreads configured to mechanically couple the housing to the scanningmicroscope.

In some embodiments, the central aperture has a diameter of about 5millimeters.

In some embodiments, the system further comprises a confocal imagingsystem comprising: a half wave plate having a first side opticallycoupled to the light source, and a second side; a polarizing beamsplitter having a first port optically coupled to the second side of thehalf wave plate, a second port optically coupled to a confocal imagingarm, and a third port optically coupled to the scanning components, andan interface that passes light having a first polarization and redirectslight having a second polarization; and a quarter wave plate having afirst side optically coupled to the scanning components, and a secondside optically coupled to the objective lens; wherein the hardwareprocessor is further programmed to: receive, from the confocal imagingarm, confocal reflectance imaging data indicative of a structure of thesample at locations at which the beam was focused; and generate a secondimage based on the confocal reflectance imaging data in parallel withthe image based on the plurality of output signals.

In some embodiments, the system further comprises a plurality of lensesconfigured to optically generate a conjugate pupil plane within theoptical path, wherein the detector is mounted within the first distanceof the conjugate pupil plane.

In some embodiments, the scanning components comprise: a firstgalvanometer optically coupled to the microscope objective; and apolygon scanner or a second galvanometer, the polygon scanner or thesecond galvanometer optically coupling the light source to the firstgalvanometer.

In accordance with some embodiments, a microscope objective is provided,comprising: a housing having a first end and a second end, the secondend comprising mounting threads; a plurality of optical componentsdefining an optical axis, the plurality of optical componentscomprising: an objective lens mounted at the first end, the objectivelens configured to collect light from a sample placed in a field of viewof the objective lens, wherein the plurality of optical componentscreate a pupil plane at a first axial distance along the optical axis atwhich rays having the same angle of incidence on the objective lens fromthe within the field of view converge at the same radial distance fromthe optical axis; a first photodetector mounted within the housing at asecond axial distance along the optical axis and offset from the opticalaxis by a first radial distance; and a second photodetector mountedwithin the housing at the second axial distance along the optical axisand offset from the optical axis by the first radial distance in adirection opposite from the first photodetector.

In some embodiments, the second axial distance is equal to the firstaxial distance.

In some embodiments, the microscope objective further comprises aphysical aperture collocated with the pupil plane, wherein the firstphotodetector and the second photodetector are mechanically coupled tothe physical aperture.

In some embodiments, the microscope objective further comprises aprinted circuit board defining a central aperture having a center,wherein the printed circuit board is mounted within the housing suchthat the center coincides with the optical axis, and wherein the firstphotodetector and the second photodetector are mechanically coupled toprinted circuit board, and electrically coupled to the first printedcircuit board.

In some embodiments, the microscope objective further comprises anamplifier electrically coupled to the printed circuit board, comprising:a first transimpedance amplifier configured to receive a first currentsignal from the first photodiode and provide a first voltage signal asan output; a second transimpedance amplifier configured to receive asecond current signal from the second photodiode and provide a secondvoltage signal as an output; a differential detection amplifierconfigured to receive the first voltage signal and the second voltagesignal, and provide a third voltage signal indicative of a differencebetween the first current signal and the second current signal as anoutput.

In some embodiments, the microscope objective further comprises, theprinted circuit board acts as a physical aperture of the microscopeobjective and is collocated with the pupil plane.

In some embodiments, the first radial distance is in a range of 2millimeters (mm) to 10 mm.

In accordance with some embodiments of the disclosed subject matter, adetection apparatus for transobjective differential epi-detection offorward scattered light is provided, the detection apparatus comprising:a housing configured to be mechanically coupled to a scanning microscopesuch that the housing is disposed along an optical path of the scanningmicroscope; a substrate having a first surface and a second surface andan aperture defined by a through-hole from the first surface to thesecond surface, the substrate mounted within the housing; a firstphotodetector mechanically coupled to the first surface of the substrateand disposed at a first distance from a side of the aperture; and asecond photodetector mechanically coupled to the first surface of thesubstrate and disposed at the first distance from an opposite side ofthe aperture from the first photodetector, such that secondphotodetector is separated from the first photodetector by the diameterof the aperture and twice the first distance.

In some embodiments, the housing is a microscope objective barrel.

In some embodiments, the substrate comprises a printed circuit board,and the first photodetector and the second photodetector aremechanically coupled to printed circuit board, and electrically coupledto the first printed circuit board..

In some embodiments, the detection apparatus further comprises anamplifier electrically coupled to the printed circuit board, theamplifier comprising: a first transimpedance amplifier configured toreceive a first current signal from the first photodiode and provide afirst voltage signal as an output; a second transimpedance amplifierconfigured to receive a second current signal from the second photodiodeand provide a second voltage signal as an output; a differentialdetection amplifier configured to receive the first voltage signal andthe second voltage signal, and provide a third voltage signal indicativeof a difference between the first current signal and the second currentsignal as an output.

In some embodiments, the first distance is in a range of 0.5 millimeters(mm) to 1 mm.

In some embodiments, the detection apparatus further comprises: a thirdphotodetector mechanically coupled to the first surface of the substrateand disposed at the first distance from a perpendicular side of theaperture to the side along which the first photodetector is disposed;and a fourth photodetector mechanically coupled to the first surface ofthe substrate and disposed at the first distance from an opposite sideof the aperture from the third photodetector.

In some embodiments, a system for differential epi-detection of forwardscattered light suitable for label free in vivo flow cytometry isprovided, the system comprising: a scanning microscope comprising: afirst light source configured to emit light at a first wavelength; asecond light source configured to emit light at a second wavelength; anoptical train defining an optical path of the scanning microscope havingan optical axis comprising: scanning components optically coupled to thelight source and configured to scan a beam from the light source acrossa surface of a sample and a microscope objective optically coupled tothe scanning components; and a detector arranged to receive lightemitted by the first light source and the second light source that hasbeen directed into a sample via the microscope objective, forwardscattered through the sample, and re-emitted from the sample on the sameside as the microscope objective, the detector comprising: at least onepair of photodiodes optically coupled to detect forward scattered lightemitted from the sample toward a first side of the microscope objectiveand a second side of the microscope objective that is opposite the firstside; an amplifier electrically coupled to the detector, comprising: adifferential amplifier configured to receive a first signal and a secondsignal from the at least one pair of photodiodes indicative of theintensity of light received at the first side of the microscopeobjective and the second side of the microscope objective at the firstwavelength, respectively, and provide a signal indicative of adifference between the first signal and the second signal as an output;and a sum amplifier configured to receive a third signal and a fourthsignal from the at least one pair of photodiodes indicative of theintensity of light received at the first side of the microscopeobjective and the second side of the microscope objective at the secondwavelength, respectively, and provide a signal indicative of a sum ofthe first signal and the second signal as an output; and at least onehardware processor that is programmed to: cause the first light sourceto emit a first beam of light toward a sample via the optical train;cause the second light source to emit a second beam of light toward asample via the optical train; cause the scanning components to scan thefirst beam of light and the second beam of light across the sample;receive, from the differential amplifier, a first plurality of outputsignals, each of the plurality of output signals indicative of astructure of the sample at a location at which the first beam wasfocused; receive, from the sum amplifier, a second plurality of outputsignals, each of the plurality of output signals indicative of anabsorption by the sample at a location at which the second beam wasfocused; and generate image data indicative of the presence of bloodcells and leukocytes in the sample based on the first plurality ofoutput signals and the second plurality of output signals.

In some embodiments, the detector is mechanically coupled to thescanning microscope along the optical path within a first distance of apupil plane of the optical train, and the detector comprises: a printedcircuit board defining a central aperture having a center configured tocoincide with the optical axis of the optical path; and the at least onepair of photodiodes comprises: a first pair of photodiodes configured toinhibit detection of light of the second wavelength, the first pair ofphotodiodes comprising: a first photodiode mechanically coupled to theprinted circuit board at a first radial distance from the center; asecond photodiode mechanically coupled to the printed circuit board atthe first radial distance from the center and on an opposite side of thecentral aperture from the first photodiode, wherein the first distanceis less than or equal to twice the first radial distance; and a secondpair of photodiodes configured to inhibit detection of light of thefirst wavelength, the second pair of photodiodes comprising: a thirdphotodiode mechanically coupled to the printed circuit board at thefirst radial distance from the center; a fourth photodiode mechanicallycoupled to the printed circuit board at the first radial distance fromthe center and on an opposite side of the central aperture from thethird photodiode.

In some embodiments, the first wavelength is in a range including nearinfrared light and excluding visible light, and the second wavelength isin a range including visible light and excluding near infrared light.

BRIEF DESCRIPTION OF THE DRAWINGS

Various objects, features, and advantages of the disclosed subjectmatter can be more fully appreciated with reference to the followingdetailed description of the disclosed subject matter when considered inconnection with the following drawings, in which like reference numeralsidentify like elements.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A1 shows an example of a backward scattering event and anassociated scattering wave vector in k-space.

FIG. 1A2 shows an example of a forward scattering event and anassociated scattering wave vector in k-space.

FIG. 1B1 shows an example of forward scattered light exiting from theface of a sample into which the light entered after multiple forwardscattering events, and the distribution of the scattered light intensityin k-space at the pupil plane.

FIG. 1B2 shows an example of less forward scattered light exiting fromthe face of a sample into which the light entered after multiple forwardscattering events due to the incidence angle of the phase object, andthe distribution of the scattered light intensity in k-space at thepupil plane.

FIG. 1B3 shows another example of forward scattered light exiting fromthe face of a sample into which the light entered after multiple forwardscattering events, and the distribution of the scattered light intensityin k-space at the pupil plane.

FIG. 2 shows an example of a system for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter.

FIG. 3 shows an example of an apparatus for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter.

FIG. 4 shows an example of rays traversing a portion of an optical pathof a simplified microscope and locations at which detectors can beplaced to facilitate differential phase contrast microscopy bytransobjective epi-detection of forward scattered light in accordancewith some embodiments of the disclosed subject matter.

FIG. 5 shows an example of rays traversing a portion of an optical pathof a simplified microscope and intersecting detectors placed near thepupil plane of the objective to facilitate differential phase contrastmicroscopy by transobjective epi-detection of forward scattered light inaccordance with some embodiments of the disclosed subject matter.

FIG. 6 shows another example of a system for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter.

FIG. 7 shows an example of a process for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter.

FIG. 8A shows multiple example systems for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light implemented in accordance with some embodiments of thedisclosed subject matter.

FIG. 8B1 shows an example of an extended pupil plane system fordifferential phase contrast microscopy by transobjective differentialepi-detection of forward scattered light implemented in accordance withsome embodiments of the disclosed subject matter.

FIG. 8B2 shows an example of a quadrature photodiode detection apparatusthat can be used in connection with an extended pupil plane system fordifferential phase contrast microscopy by transobjective differentialepi-detection of forward scattered light in accordance with someembodiments of the disclosed subject matter.

FIG. 9 shows an example of an apparatus for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light implemented in accordance with some embodiments of thedisclosed subject matter.

FIG. 10A shows an example image of an ex-vivo mouse spinal cord tissueslice generated using mechanisms described herein for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light, an image of the ex-vivo mouse spinal cordtissue slice generated using confocal reflectance microscopy techniques,and a composite combining information from both imaging modalities.

FIGS. 10B1 and 10B2 show an example image of in-vivo osteocytes in mousebone generated using mechanisms described herein for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light, and an image of the in-vivo osteocytes in mousebone generated using confocal reflectance microscopy techniques.

FIG. 11 shows examples of in vivo mouse ear skin generated usingmechanisms described herein for differential phase contrast microscopyby transobjective differential epi-detection of forward scattered light,and corresponding images generated using confocal reflectance microscopytechniques.

FIG. 12 shows an example of a portion of a signal processing techniquethat can be used to facilitate in vivo flow cytometry using signalsgenerated via differential epi-detection of forward scattered light inaccordance with some embodiments of the disclosed subject matter.

FIG. 13 shows an example of a process for in vivo flow cytometry usingsignals generated via differential epi-detection of forward scatteredlight in accordance with some embodiments of the disclosed subjectmatter.

FIG. 14 shows an example of a system for in vivo flow cytometry usingsignals generated via differential epi-detection of forward scatteredlight in accordance with some embodiments of the disclosed subjectmatter.

FIG. 15 shows examples of images of in vivo mouse ear skin generatedusing mechanisms described herein for differential epi-detection offorward scattered light, and corresponding images generated usingconfocal reflectance microscopy techniques.

DETAILED DESCRIPTION

In accordance with some embodiments of the disclosed subject matter,mechanisms (which can include systems, methods, and media) fordifferential phase contrast microscopy by transobjective detection offorward scattered light are provided.

In accordance with some embodiments of the disclosed subject matter,mechanisms described herein can utilize a detector or detectors placedat a particular radial distance from the optical axis within a plane ofan imaging system other than imaging plane to determine one or moreproperties of an object being imaged. By contrast, in conventionalimaging systems, an image of an object is formed at the image plane, andthe information at the image plane is detected by area sensor in awide-field imaging system (e.g., an array of pixels, such as a 2D arrayof CCD or CMOS pixels), by a single pixel photodetector with a pinholein a confocal scanning imaging system, by a human observer, etc. At theimaging plane of a conventional imaging system, all of the rays of lightreceived form a single point in the scene are focused at a single point,regardless of the path that the ray took. Many imaging systems, such asmicroscopes, have one or more pupil planes (sometimes referred to as thehack focal plane of a particular portion of the optics of the system) atwhich all light that entered the imaging system at a particular angleconverges at a particular radial distance from the optical axis. Asimplified example of a microscope objective pupil plane is describedbelow in connection with FIG. 4.

In scanning imaging system, scanning of the illumination beam isgenerally realized via angular tilt with the beam configured to remainstationary at the pupil plane (e.g., the axis of the tilt is about thepupil plane). As described below in connection with FIGS. 1B1 to 1B3,light from the scanning beam can enter a sample and he forward scatteredmultiple times such that it exits from same face of tissue into whichthe beam was directed. Due to refractive index variation at the focus,the forward scattered light experiences an angular tilt away from theoptical axis, and after multiple scattering, exits the sample surfacewith an uneven brightness distribution as well as an uneven angulardistribution. As described above, a tilt in the angle at which a rayintersects the image plane or the object plane transforms into a lateralshift in the pupil plane (e.g., a radial shift toward or away from theoptical axis,). Similarly, a lateral shift of the point at which a rayintersects the image plane or the object plane transforms into a tilt inthe pupil plane. In some embodiments, the mechanisms described hereincan use the multiple detectors around the pupil aperture at the pupilplane to detect a beam shift in the exiting light, and form adifferential phase-gradient image using information detected at variouslocations across the sample.

In some embodiments, mechanisms described herein can use a pair (ormultiple pairs) of photodetectors placed within the optical path of animaging system to detect the refractive index gradient along a specificorientation (or multiple specific orientations). For example, mechanismsdescribed herein can use information detected by a pair ofphotodetectors arranged at the pupil plane of a microscope objectivearound the pupil aperture of the objective at a particular radialdistance, such that the photodetectors detect light at a particularradial distance from the optical axis of the microscope objective. Insuch an example, if the photodetectors are placed on the object side ofthe aperture, the photodetectors can detect light that would otherwisebe blocked by the pupil aperture of the microscope objective, and thuscan detect information without blocking light that can be used to forman image at the image plane of the microscope.

As another example, mechanisms described herein can use informationdetected by a pair of photodetectors arranged at a conjugate of aninitial pupil plane of an imaging system (e.g., a conjugate of theobjective pupil plane), such as a secondary pupil plane, an intermediatepupil plane, an exit pupil, etc.

As yet another example, mechanisms described herein can use informationdetected by a pair of photodetectors arranged near an initial pupilplane of an imaging system (e.g., the objective pupil plane) or aconjugate of the initial pupil plane. In such an example, thephotodetectors can be placed at a radial distance from the optical axisthat is based on the distance from the pupil plane, and the angle atwhich the light diverges from the pupil plane, which can be determinedbased on the rear focal length of the lens or lenses that formed thepupil plane.

As still another example, mechanisms described herein can useinformation detected by any suitable number of photodetectors arrangedat an extended pupil plane of an imaging system (e.g., created within analternate optical path from a path of the scanning optics). In such anexample, the photodetectors can be placed at the pupil plane and cancompletely obstruct the optical path to intercept all or substantiallyall of the light at the extended pupil plane.

In some embodiments, detection of light at a particular radial distancefrom the optical axis by a pair of opposing photodetectors can bereferred to as the pupil plane differential detection (P2D2) microscopy,and does not require polarization optics, a confocal pinhole, ordescanning, yet can produces images that are free of speckle andinterference noise.

In some embodiments, mechanisms described herein can use two pairs ofphotodetectors offset by ninety degrees around the optical axis at anextended pupil plane. Additionally, in some embodiments, a beam splittercan be arranged near the pupil plane of the objective to separatescattered light from illumination light. In such embodiments, the fullpupil aperture can be used to detect the scattered light.

Image formation based on detection of light at the pupil plane does notrequire de-scanning, which decreases the light throughput, andcorresponding decreases signal to noise ratio. Additionally, pupil planedetection can occur prior to any polarization optics, scanning, andscanning lenses by occurring inside the objective lens or just after theobject lens, and can avoid noise created by such components. In someembodiments, pupil plane detection can be implemented as an add-onapparatus to an existing scanning system that can be used to provide aphase-gradient label-free image in addition to, or in lieu of, imagesprovided by the existing scanning system.

In some embodiments, the mechanisms described herein can be implementedusing an annular printed circuit board (PCB) with one or more pairs ofphotodetectors on opposing sides of a central aperture. In suchembodiments, the circuit board can be mechanically coupled to a scanningmicroscope such that the photodetectors receive light at or near thepupil plane. For example, the PCB can be mounted within a housing thatcan be configured to mechanically couple a microscope objective to themicroscope system (e.g., with female threads to accept the object andmale threads to fasten the housing to the microscope system). As anotherexample, the PCB can be integrated into a microscope objective.

In some embodiments, the aperture size of the PCB can be equal to thesize of the pupil aperture of the microscope objective. Additionally,the photodetectors can be electrically coupled to output circuitry, suchas a trans-impedance amplifier and differential-amplifier board that canbe used to generate a phase-gradient signal.

In some embodiments, mechanisms described herein can be used to generatea diffraction limited phase gradient image using photodetectors at thepupil plane. In a scanning microscope, multiple forward scattered lightexits from the same face of sample after multiple scattering with apreferred tilt angle with respect to the microscope objective, andaccordingly can be expected to arrive at a particular radial distancefrom the optical axis at the pupil plane of the microscope objectivebased on the principle that the tilt angle at the object plane istransformed into a radial shift at the pupil plane. A tilt at sampleplane causes the intensity of light at the detectors in the pupil planeto be unbalanced, producing a differential signal. If there are noscattering events at the focus of the scanning beam, the paireddetectors have the same intensity, and the differential detectionrejects background scattered light of uniform intensity, and does notreceive light scattered back at sharp angles because that light iscloser to the optical axis at the pupil plane.

In some embodiments, mechanisms described herein can be used to detectdirectly back scattered light from the sample, that has been scatteredat a particular tilt direction, as the tilt at the object plane istransformed into a lateral shift in the pupil plane which can then bedetected by photodetectors. For example, in samples that includematerial that will cause sufficient backscattering, mechanisms describedherein can detect the backscattered light that intersects the objectivelens at a particular angle, such that it is present at a particularlocation within the pupil plane.

In some embodiments, mechanisms described herein can use any suitablenumber of pairs of detectors to generate image data indicative of thesub-surface structure of a sample. For example, in some embodiments, asingle pair of photodetectors can be used to generate a singledifference signal that can be used to generate image data. As anotherexample, in some embodiments, two pairs of photodetectors offset byninety degrees can be used to generate two difference signals that canbe used to generate image data. In such an example, because the pairsare offset by ninety degrees, the two signals can be used to generatephase gradient signals at orthogonal directions. As still anotherexample, in some embodiments, multiple pairs of photodetectors can beused to generate difference signals for different wavelengths of light(e.g., using pairs of photodetectors that are sensitive to differentwavelengths).

In some embodiments, different types of detectors can be more suitablefor different types of applications. For example, a detection apparatusthat includes one or more pairs of photodetectors arranged around acentral aperture can be placed directly at or near the pupil plane ofthe microscope objective. As another example, a detection apparatus thatincludes photodetectors in a quadrature arrangement without an aperturecan be placed directly in the optical path at an extended pupil planecreated using optics to create an alternate path from the microscopeobjective. In such an example, such a detection apparatus placed at theextended pupil plane can generate a higher quality signal with lessnoise because of the larger area for light collection.

In some embodiments, mechanisms described herein can be used to generateimages indicative of the sub-surface structure of an in vivo issuesample. For example, mechanisms described herein for transobjectivedetection of forward scattered light can be used to generate DIC-likephase-gradient images of thick scattering tissue via a microscopeobjective. In some embodiments, mechanisms described herein can be usedin many applications, such as clinical and preclinical healthcare andresearch applications.

For example, conventional blood cell analysis is an invasive procedurethat requires extraction of a patient's blood, followed by ex-vivoanalysis using a flow cytometer or hemocytometer. Blood extraction isoften unpleasant and inconvenient, and is especially so inconvenient ininfants and patients who are in critical pathological condition.Accordingly, mechanisms described herein can be used to generate imagessuitable for in vivo flow cytometer based on epi-collection of forwardscattered light for label free detection of circulating blood cells andidentification of leukocytes. A flow cytometer implemented in accordancewith some embodiments of the disclosed embodiments can use real-timephase contrast and absorption contrast imaging channels to detectcirculating blood cells and identification of leukocytes.

In some embodiments, as described above, differential epi-detection offorward scattered light can be used to generate high contrast image oftissues with small refractive index variation. Additionally, a sum ofepi-detection signals of forward scattered light (e.g., at a wavelengthat which light is differentially absorbed by hemoglobin) can be used todetect local absorption. For example, a differential phase contrastchannel can be operated at a near-infrared wavelength (e.g., 925nanometers (nm), 975 nm), and a sum absorption contrast channel can beoperated at visible illumination near the hemoglobin absorption band(e.g., ˜540-600 nm). As another example, differential phase contrastchannel can be operated at a near-infrared wavelength (e.g., 925nanometers (nm), 975 nm), and a sum absorption contrast channel can beoperated at visible, ultraviolet, or infrared illumination wavelengththat is absorbed preferentially by melanin as compared to other tissueor water (one example being ˜490 nm, however many other examples existdue to the very broad absorbance spectrum of melanin) to investigate thepresence of pigmented lesions.

In some embodiments, mechanisms described herein can detect forwardscattered light at near-infrared wavelengths to generate real-timedifference signals using a high-speed analog single processing andamplification system that facilitates generation of phase contrastimaging that can be used to detect circulating blood cells in vivo.

In some embodiments, mechanisms described herein can detect forwardscattered light at visible wavelengths in a hemoglobin absorption bandto generate real-time sum signals using a high-speed analog singleprocessing and amplification system that facilitates absorption contrastimaging and can be used to label red blood cells in vivo. In someembodiments, mechanisms described herein can collect data that can beused to calculate the difference and sum signals for a particularportion of a sample simultaneously, and can utilize a combination ofphase contrast image data generated from difference signals andabsorption image data generated from sum signals that to generate highresolution phase contrast image data that can be used for blood cellcounting and absorption contrast imaging for leukocytes counting bycounting cells that are not labeled in hemoglobin absorption contrastimages as leukocytes. In some embodiments, mechanisms described hereincan include a near-infrared laser source and a suitable visible lasersource for multicolor illumination, and can include a detectionapparatus that is configured to detect forward scattered light thatexits the same side of the sample into which the light is introduced atboth wavelengths. For example, a PCB with multiple pairs ofphotodetectors can be placed at or near the pupil plane or a conjugatepupil plane, with different pairs of photodetectors configured to besensitive to light at different wavelengths (e.g., a first pair beingsensitive at the near infrared wavelength and not at the visiblewavelength, and another pair being sensitive at the visible wavelengthand not at the near infrared wavelength). As yet another example, a pairof multimode optical fibers can be attached to each side of anobjective, with one of the pair configured to detect the visible lightand the other to detect the near infrared light (e.g., via a filter onthe optical fiber, or a filter on the detector). As yet another example,light from a pair of single optical fibers placed on opposite sides ofan objective to collect forward scattered light from the sample surfacecan be separated using a dichroic filter(s). As a further example,forward scattered light emitted by the sample can be directed to pairsof near-infrared photodetectors and visible photodetectors (e.g., viaoptical fibers and a splitter, via the objective and a pupil planedetector, etc.). Note that, in some embodiments, the different colors oflight can be multiplexed using various different techniques. Forexample, multiple detectors that are sensitive to different wavelengthscan be used to detect the light simultaneously. As another example, aframe can be divided into time slots, and each light source can operatein a particular time slot, and thus the imaging can be interleaved tocapture an image with a first color using the detectors in a firstportion of the frame, and capture an image with a second color using thesame detectors in a second portion of the frame. In such an example,images can be captured at 60 frames per second, but each color can becaptured at only thirty frames per second by alternating colors ofillumination. Colors can be alternated using any suitable Scheme, suchas by frame, by line (e.g., odd lines can be capture in a first color,and even colors can be captured in another color in a first frame, andvice versa in a next frame).

Each year, millions of children die because of infection. Neonataldeaths account for 40 percent of all deaths among children under agefive. An accurate blood cell count and leukocyte count are veryimportant parameters for determining whether intervention isappropriate. However, because of impracticalities of invasive blood cellanalysis procedures, especially in infants and patients who are incritical pathological condition, frequent analysis is often deemedunjustified in many patients that may benefit from more frequentanalysis. While several noninvasive optical imaging techniques have beenproposed for in vivo blood analysis, all of them have shortcomings inpractice. RCM imaging can detect only sharp refractive index variationand suffers from speckle noise, which makes it poorly suited todetecting blood cells and blood flow. OCT can acquire cross-sectionalimages at high speed, but often has limited optical resolution. Thirdharmonic generation (THG) imaging requires high laser power that mayexceed the ANSI safety guideline, which is a particular concern in thecase of infants. Raman scattering-based imaging techniques are complex,and it is currently not possible to build a practical clinical device.Additionally, none of preceding techniques can differentiate leukocytesfrom red blood cells. In some embodiments, mechanisms described hereincan be used to implement a compact flow cytometer that generatesreal-time image data that that is indicative of the presence of bloodcells, and differentiates between leukocytes and red blood cells. Forexample, mechanisms described herein can be used to implement a handheld, low power (e.g., <10 mW laser power), and noninvasive bloodparameter analyzing device. In an example implementation described belowusing a conventional polygon-galvo scanning system, and a dataacquisition system described herein, a frame rate of at least 120 framesper second and at least 33 thousand lines per second was observed, whichis sufficient for recording blood cell flow in skin.

FIGS. 1A1 and 1A2 show examples of a backward scattering event and aforward scattering event, and associated scattering wave vectors ink-space. In FIGS. 1A1 and 1A2 k_(i) and k_(s) are wave vectors ofincident and scattered light, respectively. As shown in FIG. 1A1,scattering wave vector Δk is large and along the axial direction for thebackward scattering event. Only a sharp refractive index change alongthe direction of propagation or small scattering objects can generate alarge wave vector along the axial direction. By contrast, the scatteringwave vector Δk is relatively small and along the transverse directionfor the forward scattering event, which can be caused by larger objectswith smaller and/or more gradual changes in refractive index.Accordingly, techniques that can reject backward scattered light and/orpreferentially detect forward scattered light, such as techniquesdescribed herein for transobjective detection of forward scattered lightcan be used to visualize subtle refractive index variations in thickbiological tissues, and can be used to generate DIC-like images.

FIGS. 1B1 to 1B3 show examples of forward scattered light exiting fromthe face of a sample into which the light entered after multiple forwardscattering events, and the distribution of the scattered light intensityin k-space at the pupil plane. Principles underlying differentialphase-gradient imaging can be better understood by considering a phaseobject illuminated by a focused beam, as shown in FIGS. 1B1 to 1B3. Asshown in FIGS. 1A1 and 1A2, in accordance with the size and direction ofthe scattering wave vector Δk, the cone of forward scattered light istilted by an angle Δθ. After additional multiple scattering events, aportion of forward scattered light can re-emerge from the same face ofthe sample, as shown in FIGS. 1B1 and 1B3. The intensity distribution ofthe emergent light on the surface is dependent on the initial forwardscattering angle Δθ imposed by the phase object at the focus. Asdescribed herein, differential detection of scattered light can be usedto produce phase-gradient image data when the detectors are placed in ornear a pupil plane of an imaging system (e.g., a microscopy system, anophthalmoscope, etc.).

As described above, in addition to eliminating the need to place opticalfibers near the sample surface adjacent to the objective lens, in atelecentric scanning imaging system, the scanning beam pivots in thepupil plane and thus the pupil plane has a special relationship to thescanning beam. Beam translation at the sample plane is transformed intobeam tilting at the pupil plane, making the beam laterally stationary atthe pupil plane. When the initially forward scattered light exits fromthe face of tissue after multiple scattering, its intensityredistribution is accompanied by an angular tilt of the re-emergentlight. Just as a shift in the image plane transforms into a tilt in thepupil plane, a tilt in the image plane transforms into a shift in thepupil plane due to the two planes being a Fourier conjugate pair. FIGS.1B1 to 1B3 illustrate a beam tilt in the image plane and itscorresponding beam shift in the Fourier plane using k-space diagrams. Ak-vector in the Fourier plane corresponds to a tilt angle θ by therelation k=2 πnsin θ/λ, where n is the refractive index of the medium,and λ is the wavelength of light. The maximum tilt that the objectivelens can accept is given by the k-vector |kNA| where NA is the numericalaperture of the objective lens. A non-uniform angular distribution ofscattered light at the sample plane produces a non-uniform intensitydistribution at the pupil plane. By placing multiple detectors aroundthe pupil aperture, the intensity difference across the pupil can bemeasured. Accordingly, a differential phase-gradient image can beproduced by scanning a laser beam, as in a conventional laser scanningmicroscope, and detecting the intensity difference across the pupilusing mechanisms described herein.

FIG. 2 shows an example 100 of a system for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter. As shown, system 200 can include focusing optics (e.g.,a microscope objective), one or more scanning light sources 204;detectors 206; amplifiers and/or drivers associated with detectors 206;a hardware processor 210 configured to control operations of system 200which can include any suitable hardware processor (which can be amicroprocessor, digital signal processor, a microcontroller, an imageprocessor, a GPU, a frame grabber, etc.) or combination of hardwareprocessors; a display 212, and memory 214. Although not shown, system200 can include an input device (such as one or more buttons, amicrophone, a touchscreen, etc.) for accepting input from a user and/orfrom the environment; one or more signal generators; a communicationsystem or systems for allowing communication between hardware processor210 and other devices, such as a smartphone, a wearable computer, atablet computer, a laptop computer, a personal computer, a game console,a server, etc., via a communication link. In some embodiments, memory214 can raw data generated by detectors 206, difference signalsgenerated from the raw data, image data generated by hardware processor210, etc. Memory 214 can include a storage device (e.g., a hard disk, aBlu-ray disc, a Digital Video Disk, RAM, ROM, EEPROM, etc.) for storinga computer program for controlling hardware processor 210. In someembodiments, memory 214 can include instructions for causing hardwareprocessor 210 to execute processes associated with the mechanismsdescribed herein, such as processes described below in connection withFIGS. 7 and 13.

In some embodiments, focusing optics 202 can be any suitable optics forforming an image of a sample 216, forming a Fourier conjugate pair(e.g., at the pupil plane) of an image of sample 216, and/or projectinglight toward additional optical components that can be used to generatean image of sample at an image plane of system 200. For example,focusing optics 202 can be an objective lens, such as a microscopeobjective that includes multiple lenses. As another example, focusingoptics 202 can be an objective lens for an ophthalmoscope.

In some embodiments, scanning light source 204 can be any suitable lightsource or combination of light sources that can be configured to emitcoherent light suitable for forward-scattering imaging, and suitablecomponents for scanning the light over a portion of a sample. In someembodiments, light source 204 can emit light at any suitable wavelength,such as visible wavelengths, near infrared wavelengths, infraredwavelengths, etc. In a more particular example, light source 204 can bea diode laser that emits light centered around 648 nm. As another moreparticular example, light source 204 can be a 950 nm femto second laseror a 975 nm continuous wave laser.

In some embodiments, detectors 206 can be implemented as pairs ofphotodetectors positioned on opposing sides of an aperture that isconfigured to be positioned to coincide with an optical axis of focusingoptics 202. In some embodiments, detectors can be any suitable type ofphotodetector, such as a photodiode, a pinned photodiode, aphototransistor, an avalanche photodiode, a single photon avalanchediode (SPAD), a quantum dot photodiode, etc. In some embodiments,detectors 206 can be implemented as CMOS pixels or CCD pixels, withaccompanying driving and/or readout circuitry.

In some embodiments, amplifiers and/or drivers 208 can be any suitableamplification and/or driving circuitry that can be used to generatesignals from detectors 206. For example, drivers can be used to reset,bias, read, etc., detectors 206, and amplifiers 208 can be used togenerate a signal suitable for output to hardware processor 210.Additionally, in some embodiments, other signal processing, such as oneor more analog-to-digital circuits, frame grabbers, etc., can be used togenerate information that be processed by hardware processor 210 and/oroutput to display 212. In some embodiments, amplifiers 208 can include atransimpedance amplifier per photodetector to convert a current signalto voltage, and a differential detection amplifier to determine thedifference between the voltage signals, when detectors 206 outputcurrent signals. However, this is merely an example, and amplifiers canbe implemented using any suitable technique or combination of techniquebased on the output of detectors 206. For example, in some embodiments,detectors 206 can generate voltage signals directly (e.g., viaimplantation as CMOS active-pixel sensors). In some embodiments,amplification components can be shared. For example, in someembodiments, photodetectors can be addressable, and can be read outindividually or in pairs, and can use shared amplifier components. Notethat communication links shown in FIG. 2, such as communication linksbetween amplifiers 208 and hardware processor 210, hardware processor210 and memory 214, hardware processor 210 and memory 212, etc., can beany suitable communication links or combination of links. For example,in some embodiments, such links can be such as wired links, such aslinks (e.g., one or more serial cable links, one or more coaxial cablelinks, one or more optical fiber links, etc.), or wireless links (e.g.,Wi-Fi links, Bluetooth links, cellular links, free-space opticalcommunication links such as IrDA links, etc.). In some embodiments,detector 206 and/or amplifiers 208 can receive power from any suitablesource. For example, where a wired link is provided between detector 206and/or amplifiers 208 and hardware processor 210, power can be providedover the wired link. As another example, where no wired link is providedbetween detector 206 and/or amplifiers 208 and hardware processor 210,power can be provided by a battery or another source of power that doesnot require a wired link.

In some embodiments, display 212 can be any suitable display device(s),such as a computer monitor, a touchscreen, a television, a transparentor semitransparent display, a head mounted display, etc., and/or inputdevices and/or sensors that can be used to receive user input, such as akeyboard, a mouse, a touchscreen, a microphone, a gaze tracking system,motion sensors, etc.

FIG. 3 shows an example 300 of an apparatus for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light in accordance with some embodiments of thedisclosed subject matter. As shown in FIG. 3, apparatus 300 can includea housing 302, that can provide support for a substrate 304. In someembodiments, substrate 304 can be a printed circuit board and/or canprovide support for a printed circuit board that is mechanically coupledto substrate 304. In some embodiments, substrate 304 can be used toposition various photodetectors 306 a, 306 b, 306 c, 306 d, 308 a, 308b, and/or any other suitable photodetectors. In some embodiments, any orall of photodetectors 306 a to 306 d, 308 a, and 308 b can be used toimplement detectors 206.

As shown in FIG. 3, pairs of photodetectors 306 a to 306 d, 308 a, and308 b can be provided on opposing sides of an aperture 310 that iscentered on an optical axis 312. In some embodiments, apparatus 300 canbe configured such that in operation optical axis 312 coincides with theoptical axis of at least a portion of the optics of an imaging systemthat is used to generate data via photodetectors 306.

Although not shown, each photodetector in FIG. 3 can be electricallyconnected to one or more connection points that can be used to couplethe photodetector to output circuitry (e.g., one or more amplifiers)and/or driving circuitry (e.g., bias circuitry, timing circuitry, etc.).Additionally, in some embodiments, such circuitry can be integrated intoapparatus 300 in come embodiments. For example, one or more amplifierscan be mounted on substrate 304 and/or fabricated as part ofphotodetectors 306.

In some embodiments, different pairs of photodetectors can be configuredto be sensitive to different wavelengths of light. For example, in FIG.3, photodetectors 306 a to 306 d can be configured to be sensitive to afirst range of wavelengths (e.g., infrared, near-infrared), andphotodetectors 308 a and 308 b are sensitive to a different,non-overlapping range of wavelengths (e.g., visible light, blue light,red light, green light).

Note that although substrate 304 is shown as being substantially solid,this is merely an example, and one or more gaps can be formed insubstrate 304, such as at a position at which electrical connection arenot formed. For example, in some embodiments, the diameter of aperture310 can be adjustable, and gaps can be formed to facilitate adjustmentof the diameter such that the distance between opposing photodetectorsis adjustable while maintaining the orientation of the photodetectorswith respect to each other.

In some embodiments, the diameter of aperture 310 and/or the distancebetween opposing photodetectors (e.g., 306 a and 306 b, 306 c and 306 d,308 a, and 308 b, etc.) can be any suitable distance. For example, thewidest separation can be equal to diameter of the pupil or a conjugatepupil, as there is no light to detect outside the pupil diameter. Notethat the pupil diameter or conjugate pupil diameter can be wider ornarrower than the physical aperture diameter, and depends on the opticsof the system forming the pupil. For example, as described below inconnection with FIG. 8, optics can be included in the optical train thatcontrol a size of a conjugate pupil by creating a telescope within theoptical train. To facilitate operation of the excitation beam and/oranother imaging mode that operates through aperture 310, the diameter ofaperture 310 can be maintained above a minimum threshold. For example,aperture 310 can have a minimum diameter that is about half the diameterof the pupil near which it is situated, as a smaller diameter can nearlytotally obstruct the light source and/or light from the sample foranother imaging modality. For example, if the pupil diameter is 6 mm, adistance between opposing photodetectors can be in a range of about 3 mmto about 6 mm (e.g., each photodetector can have a radial offset ofabout 1.5 mm to about 3 mm from the radial axis), and aperture 310 cansimilarly have a diameter in a range of about 3 mm to about 6 mm,depending on how closely the photodetectors are mounted to the edge ofaperture 310. Note that pupil diameters of objectives have a wide rangeof values, and in some cases can be up to 20 mm in diameter,accordingly, aperture 310 can similarly be configured to have a diameterthat is suitable for an objective with which it is being used.

FIG. 4 shows an example of rays traversing a portion of an optical pathof a simplified microscope and locations at which detectors can beplaced to facilitate differential phase contrast microscopy bytransobjective epi-detection of forward scattered light in accordancewith some embodiments of the disclosed subject matter. As shown in FIG.4, rays that intersect the objective lens at the same angle of incidence(note that all rays are shown in the sample plane in FIG. 4), convergeat same radial distance from the optical axis at the objective lenspupil plane. The rays that intersect at 0° converge at the optical axisat the pupil plane, while rays that intersect at a steeper attack angleconverge father from the optical axis. Note that rays that diverge fromthe same point on the sample at the pupil plane converge at the samepoint at the image plane. As shown in FIG. 4, photodetectors fortransobjective differential epi-detection of forward scattered light canbe placed in various locations within an optical system, topreferentially detect rays that intersect with the objective lens in aparticular range of incidence angles, such as angles corresponding tomultiple forward-scattered light from a thick tissue sample. Potentiallocations for such detectors are illustrated in FIG. 4 in the shadedarea that corresponds roughly to the area in front of and around aphysical aperture that is often associated with the objective at thepupil plane (e.g., to block light that enters objective lens at verysteep angles from proceeding further). The precise positioning of thephotodetectors can be based on the expected incidence angles of thelight to be detected, and whether maintaining the numerical aperture ofthe optical system is desirable. For example, in a combined imagingsystem in which the central light is being used for a different imagingmodality, maintaining the numerical aperture of the optical system isgenerally desirable, and consequently placement of photodetectors infront of the physical aperture may be most appropriate if possible.Alternatively, in an imaging system in which forward scattered light isthe only imaging modality being used, detectors can be placed closer tothe optical axis of the objective lens, while still permitting the lightsource to illuminate the sample. In some embodiments, such asembodiments in which a conventional objective lens is used, placementbehind the pupil plane may be necessary due to physical inaccessibilityof the pupil plane itself. This can reduce the numerical aperture of thesystem, but as described herein, can facilitate DCI-like imaging via theobjective lens of thick tissue samples in vivo.

FIG. 5 shows an example of rays traversing a portion of an optical pathof a simplified microscope and intersecting detectors placed near thepupil plane of the objective to facilitate differential phase contrastmicroscopy by transobjective epi-detection of forward scattered light inaccordance with some embodiments of the disclosed subject matter. Asshown in FIG. 5, detectors 306 a and 306 b can be placed at the pupilplane or adjacent to but just in front of the pupil plane (i.e., on thesample side of the pupil plane), and can be supported by substrate 308,which can for example, be integrated into a microscope objective. Asshown in FIG. 5, due to the placement of the detectors at the pupilplane, the detectors collect light emitted from all points across thesample that is emitted at a particular angle with respect to theobjective lens so long as it is within the field of view of theobjective lens.

FIG. 6 shows another example 600 of a system for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light in accordance with some embodiments of thedisclosed subject matter. As shown in FIG. 6, system 600 can include alight source or multiple light sources 604, which can be used to emit abeam that is used to interrogate a sample. In some embodiments, lightsource 604 can emit a beam of light, which is reflected by a polygonscanner 606 toward a galvanometer 612 via lenses 608 and 610. In someembodiments, as polygon scanner 606 rotates, the beam is reflected at aslightly different angle, causing the beam to translate across thesurface of a sample along a first axis. Polygon scanner 606 can have anysuitable number of facets. For example, although shown with 10 facetsfor ease of observation, polygon scanner can have 36 or more facets. Insome embodiments, galvanometer 612 can rotate as polygon scanner 606rotates to cause the beam to scan along a second axis. For example,galvanometer 612 can rotate to cause the beam to translate across thesurface of a sample along a second axis each time polygon scanner 606rotates a certain number of times. Galvanometer 612 can redirect thebeam toward objective 602 via lenses 614 and 616, and can direct thebeam through an aperture in apparatus 604, which can serve as a physicalaperture of objective 602. Note that the combination of polygon scanner606 and galvanometer 612 is merely an example of scanning opticalcomponents that can be used to scan a beam across a surface of a samplevia a microscope objective in a scanning microscope, and other scanningoptical components can be used. For example, a pair of galvanometermirrors can be used to provide such scanning with one controllingscanning in a first direction and the second controlling scanning in theorthogonal direction.

In some embodiments, apparatus 604 can be an implementation of apparatus300 mounted within objective 602 at a pupil plane of objective 602. Asdescribed above, in some embodiments, polygon scanner 606 andgalvanometer 612 can cause the beam(s) of light emitted by lightsource(s) to tilt at the pupil plane of objective 602, and objective 602can focus the beam at various points of a sample, which can cause atleast a portion of the light from the beam to be forward scattered untilit is re-emitted toward objective 602. A portion of the forwardscattered light can arrive at the objective lens of objective 602 at anincident angle that causes the light to arrive at one of the detectorsof apparatus 604. In some embodiments, signals generated by detectorsassociated with apparatus 604 can be output to amplifiers and/or drivers208, and can be processed (e.g., by hardware processor 210) and/ordisplayed. Although connections are not explicitly shown in FIG. 6,hardware processor 210 can control operation of polygon scanner 606 andgalvanometer 612, for example, by providing control signals to causepolygon scanner 606 and/or galvanometer 612 to rotate.

FIG. 7 shows an example 700 of a process for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter. As shown in FIG. 7, process 700 can start at 702 byemitting light toward a portion of a sample by deflecting (or tilting) abeam toward a sample. For example, scanning components can be used todirect the beam through an objective lens at a particular location andangle such that the beam is focused at a particular position and depthof the sample.

At 704, process 700 can include collecting light using one or moredetectors located at or near the pupil plane of the objective lens,and/or at a conjugate of the objective lens pupil plane. As describedabove, photodetectors can be placed at or near the pupil plane at aparticular radial distance from the optical axis to collect light thatwas emitted from the sample (e.g., forward scattered light,backscattered light, fluoresced light, etc.) at a particular angle withrespect to the objective lens.

At 706, process 700 can determine a difference between signals fromopposing detectors. In some embodiments, any suitable techniques can beused to determine the difference between the signals from opposingdetectors. For example, current signals from opposing photodiodes can beconverted to voltage signals using transimpedance amplifiers, and theresulting voltages can be compared using a differential detectionamplifier. In some embodiments, the magnitude of the difference signalcan encode information about the composition of the sample at the pointat which the beam was aimed when the detected light was emitted. Forexample, in a more homogenous portion of the sample, forward scatteringwill occur less often, and the signals can be expected to be moresimilar. As another example, when the beam focuses on the edge of a cellmembrane, the transitions between the cell membrane and the surroundingmaterial are more likely to cause forward scattering events in onedirection than another based on the angle of incidence of the light,causing a pronounced difference in the signals between thephotodetectors (which may depend on the alignment of the blood cell andphotodetectors).

At 708, process 700 can generate image data based on scanning directionof the light source and the difference signals. In some embodiments, anysuitable technique can be used to generate image data from thedifference signals. For example, conventional linescan and framescansynchronization signals can be used to generate image frames using thedifference signal, which can be converted to image data with a digitizeror frame grabber.

FIG. 8A shows multiple example systems for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light implemented in accordance with some embodiments of thedisclosed subject matter. The system shown in FIG. 8A combinestransobjective differential epi-detection of forward scattered lightwith a confocal imaging arm that can be used simultaneously. Note thatdetectors are shown in FIG. 8A in two positions, near the pupil plane ofthe objective, and at a conjugate of the pupil plane. However, inpractice, these were implemented in the alternative, not simultaneously.The system represented in FIG. 8A is a video rate scanning platform thatincludes a 648 nm diode laser that is available from Micro Laser Systemsof Garden Grove, Calif. The laser power at the sample was limited to 5milliwatts (mW) using a half waveplate (HWP) and a polarization beamsplitter (PBS). A 36 facet polygon scanner (a Lincoln DT-36-275-040/SB12from Cambridge Technology of Bedford, Mass.) was used for fast axisscanning, and a galvanometer mirror (a model 6240 galvanometer scannerfrom Cambridge Technology) was used for slow axis scanning. A confocalimaging arm was used to compare and complement images generated usingmechanisms described herein with corresponding confocal reflectanceimages. A 25 micrometer (μm) pinhole was used to block scattered light,which is 0.6 times the airy disk size of the optical system of thesystem, and an avalanche photodiode (APD) was used to generate confocalreflectance imaging data. An Olympus 60×1.0 NA water immersion objectivelens with a 6 mm diameter aperture was used as the objective. Fourphotodiodes (SFH 2701 photodiodes available from OSRAM Licht AG ofMunich, Germany) were connected to a 15 megahertz (MHz) bandwidth custombuilt transimpedance amplifier and differential detection amplifier(DDA). Two imaging channels generated from the four photodiodes (onechannel per pair of opposing photodiodes) and a confocal reflectanceimaging channel were digitized using a 10 bit frame grabber (a SolioseAJXA Dual frame grabber available from Matrox Imaging of Dorval,Canada). The scan positions of the scanning beam on the sample wereencoded in time, such that linescan and framescan synchronizationsignals that coincided with the scan position were used by theframegrabber to generate image frames. In this configuration, thedetector was implemented as described below in connection with FIG. 9with a 5 mm aperture, and placed in a conjugate pupil plane of theobjective created by extending the imaging arm with a telescope, andmirrors. The 5 mm aperture of the ring detector reduced the excitationbeam diameter at the objective lens back pupil to 3.75 mm. The detectionpupil size at the ring detector was 8 mm, corresponding to 6 mm at thepupil of the object lens, based on the ratio of f2 to f3 shown in FIG.9. For in vivo experiments, mice were anesthetized using isoflurane andplaced on a 3-axis stage (a ROE200N stage available from SutterInstrument of Novato Calif.) with a custom 3D printed mouse holder. Allanimal experiments were performed in accordance with the institutionalguidelines for animal research. In the system represented in FIG. 8A,f1=100 mm, f2=200 mm, and f3=150 mm.

FIG. 8B1 shows an example of an extended pupil plane system fordifferential phase contrast microscopy by transobjective differentialepi-detection of forward scattered light implemented in accordance withsome embodiments of the disclosed subject matter. As shown in FIG. 8B1,an additional optical path was be created using a pellicle beam splitter(BS) to split both the illumination and the collection beam. A pair oflenses having focal lengths f3=75 mm and f4=50 mm were used to extendthe objective lens pupil plane to an extended pupil plane at which aquad photodetector (QPD) was disposed at the extended pupil plane, andobstructed the additional optical path. Since the illumination beam isable to use the full pupil aperture, and the detector is not in thepathway of the scanning beam, the system shown in FIG. 8B1 has fullresolution and full field-of-view.

FIG. 8B2 shows an example of a quadrature photodiode detection apparatusthat can be used in connection with an extended pupil plane system fordifferential phase contrast microscopy by transobjective differentialepi-detection of forward scattered light in accordance with someembodiments of the disclosed subject matter. Note that the photodiodesin the quadrature detection apparatus are large enough to capture lightfrom the entire pupil created by the lenses shown in the system of FIG.8B1

FIG. 9 shows an example of an apparatus for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light implemented in accordance with some embodiments of thedisclosed subject matter. The apparatus shown in FIG. 9 includes anannular PCB supporting four photodiode detectors arranged around a 5 mmaperture. As the exact objective pupil plane is inside the objectivelens and was inaccessible to mounting the detectors, the PCB was mountedwithin a 1″ lens tube with an adaptor for mounting the objective. Asshown in FIG. 8A, the apparatus shown in FIG. 9 was mounted fordifferent experiments at a conjugate of the pupil plane that wasgenerated by extending the imaging arm of the microscope using atelescope and mirrors (shown near the top of FIG. 8A), and behind theobjective and offset from the objective pupil plane (shown on the leftof FIG. 8A, but with the telescope removed). The photodetectors wereconnected to a custom-built differential detection amplifier, whichgenerated two real-time phase contrast imaging channels. As describedbelow in connection with FIGS. 10 and 11, techniques described hereinwere used to generate ex vivo images of mouse spinal cord and in vivoimages of mouse ear skin.

FIG. 10A shows an example image of an ex-vivo mouse spinal cord tissueslice generated using mechanisms described herein for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light, an image of the ex-vivo mouse spinal cordtissue slice generated using confocal reflectance microscopy techniques,and a composite combining information from both imaging modalities. Asshown in FIG. 10A, panel (a) shows an ex-vivo image of a mouse spinalcord tissue slice generated using the system represented in FIG. 8A inwhich the detector apparatus shown in FIG. 9 was placed at the conjugatepupil plane. FIG. 10A panel (b) is a confocal reflectance image based ondata that was generated using the confocal arm at the same time that thedata used to generate the image in panel (a) was being generated. Theimage in FIG. 10A panel (c) is a composite image created by overlayingthe confocal image in blue color on the image generated from thetransobjective differential epi-detection of forward scattered light.Contrast in these images is generated primarily from the lipid-richmyelin sheaths surrounding nerve axons. These images show that themechanisms described herein can detect lateral phase gradients andproduce a DIC-like image in a scattering tissue (spinal cord) that isdistinct from the RCM image.

FIGS. 10B1 and 10B2 show an example image of in-vivo osteocytes in mousebone generated using mechanisms described herein for differential phasecontrast microscopy by transobjective differential epi-detection offorward scattered light, and an image of the in-vivo osteocytes in mousebone generated using confocal reflectance microscopy techniques. Asshown in FIG. 10B1, the image of in-vivo osteocytes in mouse bonegenerated using the system represented in FIG. 8B1 in which the detectorapparatus shown in FIG. 8B2 was placed at the extended pupil plane. FIG.10B2 shows a confocal reflectance image based on data that was generatedusing the confocal arm shown in FIG. 8A at the same time that the dataused to generate the image in FIG. 10B1 was being generated.

FIG. 11 shows examples of in vivo mouse ear skin generated usingmechanisms described herein for differential phase contrast microscopyby transobjective differential epi-detection of forward scattered light,and corresponding images generated using confocal reflectance microscopytechniques. In a second experiment, the detector apparatus shown in FIG.9 was placed after the objective lens of the system represented in FIG.,near the objective pupil, even though the exact pupil plane isinaccessible. In this setting, the photodetectors of the detectorapparatus were shifted away from the exact objective pupil plane by ˜5mm. In this configuration, a detector apparatus can be added to anyexisting microscope without additional modification. The images in FIG.11 shows in vivo images of mouse (wild type C57BL/6) ear skin. Images inFIG. 11 panels (a) and (b) show the sebaceous gland around a hairfollicle in the reticular dermis layer, approximately 50 μm below thesurface of the mouse skin. The imaging depth of imaging based ontransobjective differential epi-detection of forward scattered lightdepends on maintaining illumination beam focus in the scattering medium.With increasing depth, contrast is reduced as the point spread functionis degraded. Accordingly, the penetration depth of techniques describedherein for transobjective differential epi-detection of forwardscattered light are similar to the penetration depth of confocalreflectance and other scanning phase gradient imaging techniques. Theimage in FIG. 11 panel (a) generated via transobjective differentialepi-detection of forward scattered light shows much more detailedcellular structures, including the nucleus of the sebaceous gland cellsand the granular structure of lipids, than the corresponding confocalreflectance image in FIG. 11 panel (b). Images in FIG. 11 panels (c) and(d) show adipocytes in the dermis layer of mouse skin, approximately 60μm below the skin surface. The image in FIG. 11 panel (c) generated viatransobjective differential epi-detection of forward scattered lightshows DIC-like morphological features while the confocal reflectanceimage in FIG. 11 panel (d) shows only the highly backscatteringstructures. FIG. 11 support that the pupil plane detector does notnecessarily need to be placed at the exact pupil plane, or even inparticularly close proximity, as the 5 nun distance from the photodiodesto the pupil plane represents a relatively large fraction of theparfocal distance of the objective.

Note that when the photodetectors are shifted away from the pupil plane,the collection beam at the detectors change both with the imaging depthand the imaging field. For example, for the 60×1.0 NA objective lensused in the system represented in FIG. 8A, if the photodiodes are 5 mmaway from the objective lens back pupil plane, the collection beamshifts by +/−0.16 mm laterally at the photodiode (due to the 5 mm offsetfrom the pupil plane) corresponding to a+/−100 μm scanning of theillumination beam at the sample plane. Note that if the detector wereplaced at the pupil plane, the beam would have only tilted at thephotodiode with no lateral shift. Further, if imaging depth is 50 μmbelow the surface, the beam diameter at the detector will be increasedby ˜0.19 mm. Thus moving the photodetectors away from the exact pupilplane results in a relatively small reduction in the illuminationfield-of-view, but otherwise has relatively little impact on the qualityof the signal acquired by the detector. Restriction of the illuminationpupil aperture by the detector apparatus reduced the resolution of theimaging system due to the detector apparatus partially blocking theoptical pathway. The numerical aperture of the imaging systemrepresented in FIG. 8A was reduced from 1.0 to 0.83 for the 60×objective. Placing detectors within the objective at the pupil plane (orotherwise in front of the physical aperture) can recover the fullnumerical aperture and full field-of-view of the microscope.

In some embodiments, the ability to place the photodetectors fortransobjective differential epi-detection of forward scattered lightdirectly after the objective lens, or inside the objective lens, canalleviate the need for descanning (e.g., as required in RCM), and caneliminate interference noise created by polarization optics, scanninglenses, and other components in the optical train. For these reasons,and other reasons described above, pupil plane detection can be acompact and inexpensive addition to a standard laser scanning microscopethat can facilitate generation DIC-like images in thick tissue samples.

FIG. 12 shows an example of a portion of a signal processing techniquethat can be used to facilitate in vivo flow cytometry using signalsgenerated via differential epi-detection of forward scattered light inaccordance with some embodiments of the disclosed subject matter. Asshown in FIG. 12, flow cytometry can be facilitated by generating adifference signal that represents phase contrast, and a sum signal thatrepresents absorption contrast. In some embodiments, any suitabletechnique or combination of technique can be used to generate the inputsignals used to generate the difference signal and sum signal. Forexample, as described above, a first pair of infrared or near infrareddetectors can be used to generate a pair of signals (IRl and IR2) thatcan be used to generate a difference signal, and a second pair ofvisible light detectors can be used to generate a pair of signals (VIS1and VIS2) that can be used to generate a sum signal.

In some embodiments, any suitable components can be used to implementthe difference and sum amplifiers. For example, IR signals can beconverted from current signals (e.g., if photodiodes are being used togenerate the signals) using a transimpedance, and differential detectionamplifier can be used to generate the difference signal (DIFF). Asanother example, VIS signals can combined as current signals, thenconverted to voltage signals and amplified for output. Alternatively,VIS signals can similarly be converted to voltage signals, and a summingamplifier can be used provide the sum signal (SUM). In some embodiments,additional signal conditioning can be applied, such as a high passfilter at each input, and a low pass filter at each output to reducenoise in the system.

FIG. 13 shows an example 1300 of a process for in vivo flow cytometryusing signals generated via differential epi-detection of forwardscattered light in accordance with some embodiments of the disclosedsubject matter. As shown in FIG. 13, process 1300 can start at 1302 byemitting light toward a portion of a sample by deflecting (or tilting) abeam toward a sample. For example, scanning components can be used todirect the beam through an objective lens at a particular location andangle such that the beam is focused at a particular position and depthof the sample.

At 1304, process 1300 can include collecting light using one or moredetectors located at or near the pupil plane of the objective lens, at aconjugate of the objective lens pupil plane, and/or near the samplesurface via optical fibers. As described above, photodetectors can beplaced at or near the pupil plane at a particular radial distance fromthe optical axis to collect light that was emitted from the sample(e.g., forward scattered light, backscattered light, fluoresced light,etc.) at a particular angle with respect to the objective lens.Additionally or alternatively, optical fibers can be placed adjacent tothe sample to collect light that has been forward scattered multipletimes and re-emitted back from the sample surface.

At 1306, process 1300 can determine a difference between signals fromopposing detectors. In some embodiments, any suitable techniques can beused to determine the difference between the signals from opposingdetectors, such as techniques described above in connection with 706 ofFIG. 7. In some embodiments, the difference signal can represent thedifference of signals detected at a particular wavelength or range ofwavelengths, such as near infrared or infrared.

At 1308, process 1300 can determine a sum between signals from opposingdetectors. In some embodiments, any suitable techniques can be used todetermine the sum between the signals from opposing detectors, such astechniques described above in connection with FIG. 12. In someembodiments, the sum signal can represent the sum of signals detected ata particular wavelength or range of wavelengths, such as visible lightor a particular relatively narrow band of visible light (e.g.,corresponding to an absorbance spectrum of hemoglobin).

At 1310, process 1300 can generate image data based on scanningdirection of the light source and the difference signals. In someembodiments, any suitable technique can be used to generate image datafrom the difference signals. For example, conventional linescan andframescan synchronization signals can be used to generate image framesusing the difference signal, which can be converted to image data with adigitizer or frame grabber.

At 1312, process 1300 can generate flow cytometry data using the imagedata. For example, process 1300 can analyze the image data to determinea red blood cell count, blood flow, a white blood cell count, etc.

FIG. 14 shows an example of a system for in vivo flow cytometry usingsignals generated via differential epi-detection of forward scatteredlight in accordance with some embodiments of the disclosed subjectmatter. The system represented in FIG. 14 combines a scanning OBMsystem, a confocal imaging arm, and a two photon fluorescence microscopysystem that can be used simultaneously. The system represented in FIG.14 was used to verify the viability of in vivo flow cytometer usingsignals generated via differential epi-detection of forward scatteredlight by recording blood flow in blood vessels of mouse ear skin. Forcomparison and verification, reflection confocal imaging system andtwo-photon imaging system were used to generate images of the samesamples. The system represented in FIG. 14 includes a 36 facet polygonscanner (a Lincoln DT-36-275-040/SB12 from Cambridge Technology ofBedford, Mass.) that was used for fast axis scanning, and a galvanometermirror (a model 6240 galvanometer scanner from Cambridge Technology)that was used for slow axis scanning. With this scanning configuration,rates of 33 thousand lines per second and up to 120 frames per secondare achievable. The field of view of the microscope was 400 μm in normalview and 200 μm in 2× zoom view. The flow cytometer had two lasersources: a 50 mW, 975 nm continuous wave diode laser from Micro LaserSystem (model L49800M-240-TE) and a 50 mW femtosecond light source at950 nm derived from a 1550 nm fiber laser (a CAZADERO laser availablefrom Calmar Laser of Palo Alto, Calif.) with soliton self-frequencyshifting and second harmonic conversion. A single mode 35 μm diameterphotonic crystal fiber (available from NKT Photonics of Birkerød,Denmark) for 1900 nm soliton generation. A frequency doubling crystal (aBiBO crystal available from Newlight Photonics of North York, Canada)was used to generate 950 nm light.

The system represented in FIG. 14 included a differential epi-detectionsystem that included two 1 mm core diameter multimode fibers (modelM59L01 available from Thorlabs of Newton, N.J.), two avalanchephotodiodes (model APD410A available from Thorlabs), and a customdifferential and sum amplifier with high-pass filters at avalanchephotodiode inputs and low-pass filters at amplifier outputs (similar tothat shown in FIG. 12). Confocal reflectance light was detected using anavalanche photodiode (model APD410A available from Thorlabs). Two-photonfluorescence light was detected using a bandpass filter centered at 532nm, a photomultiplier tube (model R7600U-200 available from HamamatsuPhotonics of Hamamatsu City, Japan) and a custom transimpedanceamplifier. The sum and difference video signals, confocal reflectancevideo signals, and two-photon video signals were digitized and acquiredusing a 10-bit frame grabber (a Solios eAJXA Dual frame grabberavailable from Matrox Imaging of Dorval, Canada). Images were capturedat 30 frames per second at full frame, 60 frames per second at halfframe, and at 17 thousand lines per second in line scan mode. 5 mW laserpower was applied at the sample.

The mice were anesthetized using isoflurane and placed on a 3-axis stage(a ROE200N stage available from Sutter Instrument of Novato Calif.) witha custom 3D printed mouse holder. A universal green fluorescent protein(GFP) mouse (beta-Actin GFP) was used to identify leukocytes forvalidation purposes.

The flow cytometer used two large-core-area multimode optical fibersattached to the two sides of objective for direct collection of forwardscattered light in epi-collection mode. In some embodiments,near-infrared and visible light from each fiber can be split by adichroic filter and detected by near-infrared and visiblephotodetectors. A high-speed analog signal processing and amplificationsystem can be used to generate the real-time difference and sum signalfor phase contrast and absorption contrast channels (e.g., as describedabove in connection with FIG. 13). For example, the difference signalcan be generated from near-infrared photodetectors and the sum signalcan be generated from visible photo detectors.

FIG. 15 shows examples of images of in vivo mouse ear skin generatedusing mechanisms described herein for differential epi-detection offorward scattered light, and corresponding images generated usingconfocal reflectance microscopy techniques. FIG. 15 panels (a) and (b)are images of a hair follicle generated using the system represented inFIG. 14 using reflection confocal images (panel (a)) and differentialepi-detection (panel (a)). The granular structure in the differentialepi-detection image of panel (b) is a sebaceous gland in upper-middermis. As shown in FIG. 15, the differential epi-detection image hasmuch higher contrast than the confocal reflectance image. The bloodcells, endothelial cells and sebaceous gland are clearly visible in thedifferential epi-detection image.

FIG. 15 panels (b) and (c) show in vivo frame-scan and line-scan imagesof blood cells flowing in a capillary of mouse ear skin. The frame-scanimage in panel (c) shows individual blood cells inside the capillaryhighlighted with arrows. Frame-scars data were taken at 30 frames persecond. The line-scan image in panel (d) was taken at 17.2 thousandlines per second across a capillary. The blood cells were flowing at 2.3mm/s through the capillary. These images confirm that differentialepi-detection imaging techniques are capable of recording individualblood cells flowing in capillaries.

FIG. 15 panels (e) and (f) show in vivo frame-scan images of blood cellsflowing in a relatively large blood vessel with GPF labeled leukocytes.Panel (e) shows a differential epi-detection image while panel (f) showsa composite image created from the differential epi-detection (gray)image and the two-photon (green) image.

In some embodiments, any suitable computer readable media can be usedfor storing instructions for performing the functions and/or processesdescribed herein. For example, in some embodiments, computer readablemedia can be transitory or non-transitory. For example, non-transitorycomputer readable media can include media such as magnetic media (suchas hard disks, floppy disks, etc.), optical media (such as compactdiscs, digital video discs, Blu-ray discs, etc.), semiconductor media(such as RAM, Flash memory, electrically programmable read only memory(EPROM), electrically erasable programmable read only memory (EEPROM),etc.), any suitable media that is not fleeting or devoid of anysemblance of permanence during transmission, and/or any suitabletangible media. As another example, transitory computer readable mediacan include signals on networks, in wires, conductors, optical fibers,circuits, any other suitable media that is fleeting and devoid of anysemblance of permanence during transmission, and/or any suitableintangible media.

It will be appreciated by those skilled in the art that while thedisclosed subject matter has been described above in connection withparticular embodiments and examples, the invention is not necessarily solimited, and that numerous other embodiments, examples, uses,modifications and departures from the embodiments, examples and uses areintended to be encompassed by the claims attached hereto. The entiredisclosure of each patent and publication cited herein is herebyincorporated by reference, as if each such patent or publication wereindividually incorporated by reference herein.

Various features and advantages of the invention are set forth in thefollowing claims.

What is claimed is:
 1. A system for differential phase contrastmicroscopy by transobjective differential epi-detection of forwardscattered light, comprising: a scanning microscope comprising: a lightsource; an optical train defining an optical path of the scanningmicroscope having an optical axis comprising: scanning componentsoptically coupled to the light source and configured to scan a beam fromthe light source across a surface of a sample; and a microscopeobjective optically coupled to the scanning components; and a detectormechanically coupled to the scanning microscope along the optical pathwithin a first distance of a pupil plane of the optical train, thedetector comprising: a printed circuit board defining a central aperturehaving a center configured to coincide with the optical axis of theoptical path; a first photodiode mechanically coupled to the printedcircuit board at a first radial distance from the center; and a secondphotodiode mechanically coupled to the printed circuit board at thefirst radial distance from the center and on an opposite side of thecentral aperture from the first photodiode, wherein the first distanceis less than or equal to twice the first radial distance; an amplifierelectrically coupled to the detector, comprising: a first transimpedanceamplifier configured to receive a first current signal from the firstphotodiode and provide a first voltage signal as an output; a secondtransimpedance amplifier configured to receive a second current signalfrom the second photodiode and provide a second voltage signal as anoutput; a differential detection amplifier configured to receive thefirst voltage signal and the second voltage signal, and provide a thirdvoltage signal indicative of a difference between the first currentsignal and the second current signal as an output; and at least onehardware processor that is programmed to: cause the light source to emita beam of light toward the sample via the optical train; cause thescanning components to scan the beam of light across the sample;receive, from the differential detection amplifier, a plurality ofoutput signals, each of the plurality of output signals indicative of astructure of the sample at location at which the beam was focused;generate an image based on the plurality of output signals; and causethe image to be presented using a display.
 2. The system of claim 1,wherein the detector is integrated within the microscope objective. 3.The system of claim 1, wherein the detector is mounted between themicroscope objective and the second plurality of lenses, the detectorfurther comprising: a housing supporting the printed circuit board;first threads configured to receive the microscope objective; and secondthreads configured to mechanically couple the housing to the scanningmicroscope.
 4. The system of claim 1, wherein the central aperture has adiameter of about 5 millimeters.
 5. The system of claim 1, furthercomprising a confocal imaging system comprising: a half wave platehaving a first side optically coupled to the light source, and a secondside; a polarizing beam splitter having a first port optically coupledto the second side of the half wave plate, a second port opticallycoupled to a confocal imaging arm, and a third port optically coupled tothe scanning components, and an interface that passes light having afirst polarization and redirects light having a second polarization; anda quarter wave plate having a first side optically coupled to thescanning components, and a second side optically coupled to theobjective lens; wherein the hardware processor is further programmed to:receive, from the confocal imaging arm, confocal reflectance imagingdata indicative of a structure of the sample at locations at which thebeam was focused; and generate a second image based on the confocalreflectance imaging data in parallel with the image based on theplurality of output signals.
 6. The system of claim 1, furthercomprising a plurality of lenses configured to optically generate aconjugate pupil plane within the optical path, wherein the detector ismounted within the first distance of the conjugate pupil plane.
 7. Thesystem of claim 1, wherein the scanning components comprise: a firstgalvanometer optically coupled to the microscope objective; and apolygon scanner or a second galvanometer, the polygon scanner or thesecond galvanometer optically coupling the light source to the firstgalvanometer.
 8. A microscope objective, comprising: a housing having afirst end and a second end, the second end comprising mounting threads;a plurality of optical components defining an optical axis, theplurality of optical components comprising: an objective lens mounted atthe first end, the objective lens configured to collect light from asample placed in a field of view of the objective lens, wherein theplurality of optical components create a pupil plane at a first axialdistance along the optical axis at which rays having the same angle ofincidence on the objective lens from the within the field of viewconverge at the same radial distance from the optical axis; a firstphotodetector mounted within the housing at a second axial distancealong the optical axis and offset from the optical axis by a firstradial distance; and a second photodetector mounted within the housingat the second axial distance along the optical axis and offset from theoptical axis by the first radial distance in a direction opposite fromthe first photodetector.
 9. The microscope objective of claim 8, whereinthe second axial distance is equal to the first axial distance.
 10. Themicroscope objective of claim 8, further comprising a physical aperturecollocated with the pupil plane, wherein the first photodetector and thesecond photodetector are mechanically coupled to the physical aperture.11. The microscope objective of claim 8, further comprising a printedcircuit board defining a central aperture having a center, wherein theprinted circuit board is mounted within the housing such that the centercoincides with the optical axis, and wherein the first photodetector andthe second photodetector are mechanically coupled to printed circuitboard, and electrically coupled to the first printed circuit board. 12.The microscope objective of claim 11, further comprising an amplifierelectrically coupled to the printed circuit board, comprising: a firsttransimpedance amplifier configured to receive a first current signalfrom the first photodiode and provide a first voltage signal as anoutput; a second transimpedance amplifier configured to receive a secondcurrent signal from the second photodiode and provide a second voltagesignal as an output; a differential detection amplifier configured toreceive the first voltage signal and the second voltage signal, andprovide a third voltage signal indicative of a difference between thefirst current signal and the second current signal as an output.
 13. Themicroscope objective of claim 11, wherein the printed circuit board actsas a physical aperture of the microscope objective and is collocatedwith the pupil plane.
 14. The microscope objective of claim 11, whereinthe first radial distance is in a range of 2 millimeters (mm) to 10 mm.15. A detection apparatus for differential phase contrast microscopy bytransobjective differential epi-detection of forward scattered light,comprising: a housing configured to be mechanically coupled to ascanning microscope such that the housing is disposed along an opticalpath of the scanning microscope; a substrate having a first surface anda second surface and an aperture defined by a through-hole from thefirst surface to the second surface, the substrate mounted within thehousing; a first photodetector mechanically coupled to the first surfaceof the substrate and disposed at a first distance from a side of theaperture; and a second photodetector mechanically coupled to the firstsurface of the substrate and disposed at the first distance from anopposite side of the aperture from the first photodetector, such thatsecond photodetector is separated from the first photodetector by thediameter of the aperture and twice the first distance.
 16. The detectionapparatus of claim 15, wherein the housing is a microscope objectivebarrel.
 17. The detection apparatus of claim 15, wherein the substratecomprises a printed circuit board, and wherein the first photodetectorand the second photodetector are mechanically coupled to printed circuitboard, and electrically coupled to the first printed circuit board.. 18.The detection apparatus of claim 17, further comprising an amplifierelectrically coupled to the printed circuit board, the amplifiercomprising: a first transimpedance amplifier configured to receive afirst current signal from the first photodiode and provide a firstvoltage signal as an output; a second transimpedance amplifierconfigured to receive a second current signal from the second photodiodeand provide a second voltage signal as an output; a differentialdetection amplifier configured to receive the first voltage signal andthe second voltage signal, and provide a third voltage signal indicativeof a difference between the first current signal and the second currentsignal as an output.
 19. The detection apparatus of claim 17, whereinthe first distance is in a range of 0.5 millimeters (mm) to 1 mm. 20.The detection apparatus of claim 15, further comprising: a thirdphotodetector mechanically coupled to the first surface of the substrateand disposed at the first distance from a perpendicular side of theaperture to the side along which the first photodetector is disposed;and a fourth photodetector mechanically coupled to the first surface ofthe substrate and disposed at the first distance from an opposite sideof the aperture from the third photodetector.
 21. A system fordifferential epi-detection of forward scattered light suitable for labelfree in vivo flow cytometry, comprising: a scanning microscopecomprising: a first light source configured to emit light at a firstwavelength; a second light source configured to emit light at a secondwavelength; an optical train defining an optical path of the scanningmicroscope having an optical axis comprising: scanning componentsoptically coupled to the light source and configured to scan a beam fromthe light source across a surface of a sample; and a microscopeobjective optically coupled to the scanning optical components; and adetector arranged to receive light emitted by the first light source andthe second light source that has been directed into a sample via themicroscope objective, forward scattered through the sample, andre-emitted from the sample on the same side as the microscope objective,the detector comprising: at least one pair of photodiodes opticallycoupled to detect forward scattered light emitted from the sample towarda first side of the microscope objective and a second side of themicroscope objective that is opposite the first side; an amplifierelectrically coupled to the detector, comprising: a differentialamplifier configured to receive a first signal and a second signal fromthe at least one pair of photodiodes indicative of the intensity oflight received at the first side of the microscope objective and thesecond side of the microscope objective at the first wavelength,respectively, and provide a signal indicative of a difference betweenthe first signal and the second signal as an output; and a sum amplifierconfigured to receive a third signal and a fourth signal from the atleast one pair of photodiodes indicative of the intensity of lightreceived at the first side of the microscope objective and the secondside of the microscope objective at the second wavelength, respectively,and provide a signal indicative of a sum of the first signal and thesecond signal as an output; and at least one hardware processor that isprogrammed to: cause the first light source to emit a first beam oflight toward a sample via the optical train; cause the second lightsource to emit a second beam of light toward a sample via the opticaltrain; cause the scanning components to scan the first beam of light andthe second beam of light across the sample; receive, from thedifferential amplifier, a first plurality of output signals, each of theplurality of output signals indicative of a structure of the sample at alocation at which the first beam was focused; receive, from the sumamplifier, a second plurality of output signals, each of the pluralityof output signals indicative of an absorption by the sample at alocation at which the second beam was focused; and generate image dataindicative of the presence of blood cells and leukocytes in the samplebased on the first plurality of output signals and the second pluralityof output signals.
 22. The system of claim 21, wherein the detector ismechanically coupled to the scanning microscope along the optical pathwithin a first distance of a pupil plane of the optical train, and thedetector comprises: a printed circuit board defining a central aperturehaving a center configured to coincide with the optical axis of theoptical path; and the at least one pair of photodiodes comprises: afirst pair of photodiodes configured to inhibit detection of light ofthe second wavelength, the first pair of photodiodes comprising: a firstphotodiode mechanically coupled to the printed circuit board at a firstradial distance from the center; a second photodiode mechanicallycoupled to the printed circuit board at the first radial distance fromthe center and on an opposite side of the central aperture from thefirst photodiode, wherein the first distance is less than or equal totwice the first radial distance; and a second pair of photodiodesconfigured to inhibit detection of light of the first wavelength, thesecond pair of photodiodes comprising: a third photodiode mechanicallycoupled to the printed circuit board at the first radial distance fromthe center; a fourth photodiode mechanically coupled to the printedcircuit board at the first radial distance from the center and on anopposite side of the central aperture from the third photodiode.
 23. Thesystem of claim 21, wherein the first wavelength is in a range includingnear infrared light and excluding visible light, and the secondwavelength is in a range including visible light and excluding nearinfrared light.